Purification and characterization of extracellular aspartic proteinase of Candida albicans

An extracellular proteinase of Candida albicans was purified by a combination of 0-75% ammonium sulfate precipitation, DEAE Sepharose Fast Flow ion exchange chromatography, and Sephacryl S-200 HR molecular sieve chromatography. Its molecular weight was approximately 41 kDa on SDS-PAGE and isoelectric point was 4.4. The enzyme was inhibited by pepstatin A. Optimum enzyme activity ranged from pH 2.0 to 3.5 with its maximum at pH 2.5 and a temperature of 45 degrees C. The addition of divalent cations, Ca2+, Zn2+ and Mg2+, resulted in no significant inhibition of enzymatic activity. However, some inhibitory effects were observed by Fe2+, Ag2+ and Cu2+. With BSA as substrate, an apparent K-m was determined to be 7x10(-7) M and K-i, using pepstatin A as an inhibitor, was 8.05x10(-8) M. N-terminal amino acid sequence was QAVPVTLX-NEQ. Degradation of BSA and fibronectin was shown but not collagen, hemoglobin, immunoglobulin G, or lysozyme. The enzyme preferred peptides with Glu and Leu at the P-1 position, but the enzyme activity was highly reduced when the P-2 position was Phe or Pro. This enzyme showed antigenicity against sera of patients with candidiasis.